This invention relates to devices and methods for performing active, multi-step sample preparation and molecular diagnostic analysis of biological materials. More particularly, it relates to integrated, compact, portable devices for self-contained sample to answer systems. Specifically, this invention relates to a device and method for performing multi-step sample preparation and assay on either two or even a single microchip. Examples of applications for this integrated system include food and/or quality monitoring, diagnosis of infectious diseases and cancers, bone marrow plastesis (e.g., stem cell separation and analysis), and genetics-based identification of individuals for forensic purposes.
The following description provides a summary of information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to the invention.
Generally, analysis of biological-derived sample materials cannot occur until the sample is processed through numerous pre-analysis steps. Often, the preparation process is time consuming and laborious. For example, many immuno and molecular-biological diagnostic assays on clinical samples, such as blood or tissue cells, require separation of the molecules of interest from the crude sample by disrupting or lysing the cells to release such molecules including proteins and nucleic acids (i.e., DNA and RNA) of interest, followed by purification of such proteins and/or nucleic acids. Only after performing processing steps can analysis of the molecules of interest begin. Additionally, protocols used for the actual analysis of the samples require numerous more steps before useful data is obtained.
For example, charged and uncharged microparticles in solution (such as cellular material or crude extracts of protein or nucleic acids thereof) may be separated by dielectrophoresis. On a microscale, dielectrophoresis can be performed using a glass slide-based device having exposed, i.e., naked, interdigitated electrodes plated on the surface of the slide and having a flow chamber with a volume of several hundred microliters. With such a device, cells, proteins, and nucleic acids can be separated based on their respective dielectric properties by using separation buffers having appropriate conductivity and an AC signal with a suitable amplitude and frequency. Such devices, however, have several problems including the nonspecific binding of both separated and unseparated cells to exposed portions of the glass surface and the electrodes. Such devices are also problematic in that the flow chamber volume (several hundred microliters) is so large that thermal convection can disturb and push materials such as cells and large molecules initially attracted to and retained by the electrodes off of the electrodes. Additionally, undesired cells and molecules are not easily washed off the surface without disturbing and loosing the desired cells as such cells and molecules can interfere with fluidic flow and, hence, block the flow during wash steps.
Conventional methods to disrupt whole cells for the release of proteins and nucleic acids have employed the use of a series of high voltage DC pulses in a macrodevice, as opposed to a microchip-based device. Such conventional electronic lysis techniques have several problems. For example, some commercial macro-devices use lysis conditions that do not release high molecular weight (larger than 20 Kb) nucleic acids because the high molecular weight molecules can not fit through pores created in the cell membranes using such methods. Additionally, released nucleic acids are often lost due to their non-specific binding to the surface of the lysis chamber. Such loss of material, especially when molecules of interest are in low concentration, is further compounded by the fact that the dielectrophoretic cell separation macro-device systems are stand alone systems allowing for loss of sample in the transfer of material from one device to the other as sample preparation is carried forward.
Processing of the crude lysate often requires chemical reactions to remove undesired cellular components from the specifically desired ones. These reactions typically include subjecting the lysate to enzymatic reactions such as proteinase K and restriction enzymes or nucleases. Processing can also include enhancing the presence of desired molecules, particularly nucleic acids, by performing amplification reactions such as by strand displacement amplification (SDA) or polymerase chain reaction (PCR) methodologies. These reactions are also carried out in stand-alone processes. Only after these sample preparation and processing steps can assaying for data retrieval begin. Because of the numerous steps between sample collection and assay, many a technique is limited in its application by a lack of sensitivity, specificity, or reproducibility.
Attempts have been made to use dielectrophoresis to separate and identify whole cells. For example, U.S. Pat. No. 4,326,934 to Pohl discloses a method and apparatus for cell classification by continuous dielectrophoresis. With such method cells are separated by making use of both the positive and negative dielectrophoretic movement of cell particles. Separated cells are allowed to be characterized and/or classified by viewing the characteristic deflection distance of cells moving through the positive and negative electrodes.
In another example, U.S. Pat. No. 5,344,535 to Belts et al. discloses a method and apparatus for the characterization of micro-organisms and other particles by dielectrophoresis. In this system, cells are characterized by matching their signature dielectrophoretic collection rates.
In yet another example, U.S. Pat. No. 5,569,367 to Belts et al. discloses a method and apparatus for separating a mixture of cells using a pair of energized interdigitated electrodes comprised of interweaved grid-like structures arranged to obstruct flow of cells through the apparatus and cause differentiation of cell types into fractions by applying a non-uniform alternating field.
In addition, other attempts have been made to combine certain processing steps or substeps together. For example, various microrobotic systems have been proposed for preparing arrays of DNA probes on a support material. Beattie et al., disclose in xe2x80x9cThe 1992 San Diego Conference: Genetic Recognitionxe2x80x9d, November, 1992, use of a microrobotic system to deposit micro-droplets containing specific DNA sequences into individual microfabricated sample wells on a glass substrate.
Various other attempts have been made to describe integrated systems formed on a single chip or substrate, wherein multiple steps of an overall sample preparation and diagnostic system would be included. A. Manz et al., in xe2x80x9cMiniaturized Total Chemical Analysis System: A Novel Concept For Chemical Sensingxe2x80x9d, Sensors And Actuators, B1(1990), pp. 244-248, describe a xe2x80x98total chemical analysis systemxe2x80x99 (TAS) that comprises a modular construction of a miniaturized TAS. In that system, sample transport, chemical reactions, chromatographic separations and detection were to be automatically carried out.
Yet another proposed integrated system by Stapleton, U.S. Pat. No. 5,451,500, a system for automated detection of target nucleic acid sequences is described. In this system multiple biological samples are individually incorporated into matrices containing carriers in a two-dimensional format.
Various multiple electrode systems are also disclosed which purport to perform multiple aspects of biological sample preparation or analysis. Pace, U.S. Pat. No. 4,908,112, entitled xe2x80x9cSilicon Semiconductor Wafer for Analyzing Micronic Biological Samplesxe2x80x9d describes an analytical separation device in which a capillary-sized conduit is formed by a channel in a semiconductor device, wherein electrodes are positioned in the channel to activate motion of liquids through the conduit. Additionally, Soane et al., in U.S. Pat. No. 5,126,022, entitled xe2x80x9cMethod and Device for Moving Molecules by the Application of a Plurality of Electrical Fieldsxe2x80x9d, describes a system by which materials are moved through trenches by application of electric potentials to electrodes in which selected components may be guided to various trenches filled with antigen-antibodies reactive with given charged particles being moved in the medium or moved into contact with complementary components, dyes, fluorescent tags, radiolabels, enzyme-specific tags or other types of chemicals for any number of purposes such as various transformations which are either physical or chemical in nature. Further, Clark, et al. in U.S. Pat. No. 5,194,133, entitled xe2x80x9cSensor Devicesxe2x80x9d, discloses a sensor device for the analysis of a sample fluid which includes a substrate having a surface in which is formed an elongate micro-machined channel containing a material, such as starch, agarose, alginate, carrageenan or polyacrylamide polymer gel, for causing separation of the sample fluid as the fluid passes along the channel. The biological material may comprise, for example, a binding protein, an antibody, a lectin, an enzyme, a sequence of enzymes, or a lipid.
Various devices for eluting DNA from various surfaces are known. For example, Shukla U.S. Pat. No. 5,340,449, entitled xe2x80x9cApparatus for Electroelutionxe2x80x9d describes a system and method for the elution of macromolecules such as proteins and nucleic acids from solid phase matrix materials such as polyacrylamide, agarose and membranes such as PVDF in an electric field. Materials are eluted from the solid phase into a volume defined in part by molecular weight cut-off membranes. Also, Okano, et al. in U.S. Pat. No. 5,434,049, entitled xe2x80x9cSeparation of Polynucleotides Using Supports Having a Plurality of Electrode-Containing Cellsxe2x80x9d discloses a method for detecting a plurality of target polynucleotides in a sample, the method including the step of applying a potential to individual chambers so as to serve as electrodes to elute captured target polynucleotides, the eluted material is then available for collection.
Other devices for performing nucleic acid diagnosis have been designed wherein at least two reaction chambers are necessary for carryout the sample preparation and analysis such as R. Lipshutz, et al., entitled xe2x80x9cIntegrated Nucleic Acid Diagnostic Devicexe2x80x9d (U.S. Pat. No. 5,856,174) and R. Anderson, et al., entitled xe2x80x9cIntegrated Nucleic Acid Diagnostic Devicexe2x80x9d, (U.S. Pat. No. 5,922,591).
Still other achievements have been made toward partial integration of a complete sample handling system such as P. Wilding, et al., xe2x80x9cIntegrated cell isolation and PCR analysis using silicon microfilter-chambers,xe2x80x9d Anal. Biochem. 257, pp. 95-100, 1998; and P. C. H. Li and D. J. Harrison, xe2x80x9cTransport, manipulation, and reaction of biological cells on-chip using electrokinetic effects,xe2x80x9d Anal. Chem., 69, pp. 1564-1568, 1997.
Still others have attempted to integrate chemical reactions with detection such as M. A. Burns, et al., xe2x80x9cAn integrated nanoliter DNA analysis device,xe2x80x9d Science, 282, pp. 484-487, 1998; S. C. Jacobson and J. M. Ramsey, xe2x80x9cIntegrated microdevice for DNA restriction fragment analysis,xe2x80x9d Anal. Chem., 68, pp. 720-723, 1996; L. C. Waters, et al., xe2x80x9cMicrochip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing,xe2x80x9d Anal. Chem., 70, pp. 158-162, 1998; and A. T. Woolley, et al., xe2x80x9cFunctional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device,xe2x80x9d Anal. Chem., 68, pp. 4081-4086, 1996.
Generally, as is understandable from the forgoing examples, systems and methods have been described that do not fully provide for a completely integrated self-contained sample to answer system that uses electronically active microchips. Moreover, numerous of the described systems are extremely labor and time intensive requiring multiple steps and human intervention either during the process or between processes which together are suboptimal allowing for loss of sample, contamination, and operator error. Further, the use of multiple processing steps using multiple machines or complicated robotic systems for performing the individual processes is often prohibitive except for the largest laboratories, both in terms of the expense and physical space requirements. For the reasons stated above, these techniques are limited and lacking. They are not easily combined to form a system that can carry out a complete self-contained integrated diagnostic assay, particularly assays for data retrieval for nucleic acids and protein-derived information, on a single electronically addressable microchip. Despite the long-recognized need for such an integrated system without a complicated fluidics and inadequate valve systems, no satisfactory solution has previously been proposed. There is therefore a continuing need for methods and devices which lead to improved dielectrophoretic separation of biological cells as well as improved biological stability of the separated cells and further a continuing need for methods and devices which improve cell preparation and analysis, and which are capable of integrating cell separation, preparation, purification, and analysis in a single self-contained system without complicated fluidics.
Accordingly, provided herein is an integrated, portable system and device for performing active, integrated multi-step sample preparation and molecular diagnostic analysis of biological samples using a minimal number of electronically addressable microchips.
In a preferred embodiment, the system and device has a single flow cell element for carrying out the separation, preparation, and analysis of preselected eukaryotic and/or prokaryotic cells from a mixed population of cells. This single flow cell element includes therein an electronically addressable microchip having a grid of individual electrodes. The grid can contain any number of individual electrodes and be fabricated on a silicon wafer as is well understood in the electronic arts. Generally, such a grid can comprise anywhere from 4 to 25 to 100 or even 10,000 or more individual electrodes. This single flow cell embodiment contemplates that the flow cell encompassing the electronic grid is able to achieve simple fluidic manipulation of the cells for their respective separation, lysis, clean-up of cell-derived materials of interest, and analysis of such materials. Preselected eukaryotic and prokaryotic cells may be any type (e.g., blood cells, cancer cells, stem cells, bacteria, etc.) and obtained from any source (e.g., animal, biopsy, blood, organs, forensic samples, stool, water, etc.). Specific examples of cells that can be separated from, for example, whole blood include Escherichia coli, Salmonella typhimurium, Staphylococuus epidermus, Micrococcus lysodeikticus, and cultured cervical carcinoma cells.
In an alternative embodiment, the device of the invention may include at least one additional flow chamber for carrying out either a variety of sample preparation steps or for analysis of prepared sample. For example, in one alternative embodiment, the system comprises a first and second flow cell chambers each having an electronically addressable microchip. In this alternative embodiment, the first chamber may be used to separate and lyse cells as well as perform sample preparation steps, while the second chamber is used to analyze the sample materials directly on the second flow cell microchip.
In another alternative embodiment comprising at least two flow cells, the first flow cell has no electronically addressable grid but does have at least a heating element attached thereto that can be used to adjust the temperature of materials within said cell. The second flow cell is contemplated to have an electronically addressable microarray for use in performing analysis of the sample materials.
Regardless of the embodiment utilized, the system of the invention provides the ability to (1) separate eukaryotic cell types from one another as well as eukaryotic cell types from prokarotic cell types, (2) directly process the sample materials from a crude state to a more refined state, and (3) directly analyze the sample materials on the microchip grid. Such an ability is possible by the novel use of electronic biasing at one level of voltage in the form of a dielectric current to cause dielectrophoresis of cells, followed by an increase in voltage to lyse captured cells, followed in turn by changing the manner of biasing from an alternating current mode to direct current mode for the addressing of specific electrodes on the arrays of the flow cell(s) to cause the transport of molecules of interest for capture/hybridization to probes previously bound to the electrode array. The system of the invention further contemplates that other appropriate sample preparation reagents may be transported to and away from the flow cell(s) by simplified arrangement of tubing and solenoid operated valves and pump. Additionally, in embodiments having a first flow chamber that is without a microchip, the system contemplates the ability to directly lyse the cells in the sample and analyze materials of interest without a need for separating the cell types. In such embodiment, the flow cell has a heating element that can be used to raise the temperature for direct lysis of the cells in the sample. Following such lysis, sample preparation steps such as protease treatment or nucleic acid amplification may be performed followed by transporting the amplified species to the second flow cell containing the electronically addressable microchip.
In another embodiment, the integrated system contemplates the direct processing of cells selectively separated or lysed in the first chamber to obtain any materials of interest from such cells. Such processing is generally directed to the isolation and purification of proteins and nucleic acids from cellular contaminants. Numerous techniques can be performed in the preparation of molecules of interest including, but not limited to, enzymatic treatment using protease K to remove proteinaceous materials from nucleic acids of interest, enzymatic treatment using nucleases to remove nucleic acids from proteins, digestive residue adsorption, nucleic acid amplification (e.g., by PCR and SDA), in situ buffer exchange and binding of antibodies or other protein-protein binding reactions such as receptor-ligand or enzyme-substrate for binding to proteins of interest.
In another embodiment, analysis of prepared sample materials can comprise any number of preselected hybridization formats. In a preferred format, nucleic acids of interest are hybridized selectively through an electronically directed process as is known to those skilled in the art of electronically addressable microchips. Such hybridization formats comprise binding of nucleic acids (RNA, DNA, pNA) to probes anchored to the microarray. Other formats contemplated for use with the system include selective capture of proteins of interest such as by antibody or other protein binding probes attached to the electronic grid. These can include other protein-protein interactions such as receptor-ligand and enzyme-substrate binding.
In still other embodiments, the device contemplates elements (e.g. buffer vials, tubing, miniature solenoid valves, and at least one pump) for carrying and transporting samples, buffers, enzymes and reagents to and from said flow cell(s). Additionally, yet other embodiments are provided for detecting the analytes and molecules of interest being assayed. In a preferred embodiment, such elements include a battery operated diode laser (preferably having a wavelength of 635 nm), and a CCD camera coupled with filters and zoom lens for astronomy of the individual electrodes of the microchip grid(s) of the first and/or second flow cell chamber(s). Still other methods of detection are also contemplated not requiring illumination with a light emitting device such as direct electrochemical detection as is well known by those in the art of such detection as described in P. Ropp and H. Holden Thorp, Chemistry and Biology, 1999, Vol. 6, No. 9, pp. 599-605. Each of these electronic components are further contemplated to be coordinated through a computer and appropriate programming software as is well understood by those skilled in the electronic arts.